ANALYSIS OF HIV-1 GAG-MEDIATED ASSEMBLY

Project: Research project

Project Details

Description

Retroviruses are important etiological agents of human disease most
notable causing acquired immune deficiency syndrome (AIDS). The
molecular events by which progeny retroviruses are produced is poorly
understood, though fundamental to the virus replication cycle. In this
application for a Physician-Scientist Award, a research training program
is proposed to address retrovirus assembly, while preparing the P.I. for
an independent research career. The retrovirus to be studied in this
program is the AIDS virus HIV.

The HIV-1 Gag protein directs retroviral assembly (budding), although its
molecular mechanism remains obscure. Since budding is essential for the
production of infectious virions, its disruption is a potential target
for antiretroviral therapy. In the Phase I research plan, three separate
projects are proposed. First, an extensive deletion analysis will be
performed to map the essential and nonessential regions of HIV-1 Gag.
Those portions which are identified as critical to budding will be
further characterized. Specifically, the domain of Gag which associates
with the cytoplasmic membrane will be mapped to allow further study of
the Gag-membrane interaction, which is believed to be the first step in
retroviral assembly. Once membrane-associated, the Gag proteins form
aggregates which provide the nucleation site for assembly. The specific
domains of the Gag protein which are responsible for these Gag-Gag
interactions will be identified and characterized.

The second project will focus on the development of an in vitro assay to
study how the HIV-1 Gag protein interacts with the cytoplasmic membrane.
Using a cell-free system similar to. that used to identify the membrane
receptor for p60(src) (Resh and Ling, 1990), in vitro-translated Gag
proteins will be incubated with osmotically-lysed cell membrane
preparations and subjected to cell fractionation studies. If specific
Gag-membrane associations can be detected, we will proceed with
experiments aimed at the identification of the putative cytoplasmic
receptor for HIV-1 Gag.

The third aim of this proposal is a pilot project to study in vivo the
cellular factors involved in HIV-1 Gag-mediated assembly. Previous
studies of Gag function in yeast have demonstrated that retroviral Gag
proteins associate with the cell membrane, but the budding process is
inhibited, likely by the rigid cell wall. We propose to study Gag
function in Dictyostelium, a wall-less lower eukaryote with a
well-established genetic system. If HIV-1 Gag can mediate budding in
Dictyostelium, powerful genetic analyses can be performed to elucidate
the role of cell-encoded proteins in the retroviral assembly apparatus.

Phase II is planned as an intense laboratory experience which will
develop the candidate's independent research career. A general research
plan is included for Phase II, which will be based on the results of the
experiments planned in the Phase I research program.
StatusFinished
Effective start/end date6/1/935/31/94

Funding

  • National Institute of Allergy and Infectious Diseases

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